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Objective: To detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads., Methods: The sensitivity of 2 techniques, monoclonal antibody specific immobilization of platelet antigen (MAIPA) and Luminex bead assay, was compared using 12 twofold-serial dilutions (from neat to 1 in 2048) of an anti-HPA-1a WHO international standard. The specificity of Luminex assay to identify anti-HPA-1a and -1b antibodies was assessed using 8 negative or positive controls and 36 blinded samples provided by WHO Platelet Workshop., Results: The sensitivity of MAIPA and Luminex bead assay to detect anti-HPA-1a was dilution 1/64 (i.e. 1.56 IU/ml) and far more than dilution 1/2048 (i.e. 0.049 IU/mL), respectively. The Luminex bead assay could specifically identify negative and positive controls of anti-HPA-1a and -1b. All results of 36 blinded samples by Luminex assay were accordant to reference results except one sample which contained high concentration antithetical antibody and resulted in false positive of anti-HPA-1b. Cross-reactivity was also not observed with the samples containing HLA, ABO or other platelet antibodies., Conclusion: The Luminex beads coupled with recombinant GPIIIa fragments can be used to detect HPA-1 system antibodies with sufficient sensitivity and specificity, that is suitable for the detection of platelet alloantibodies in clinical alloimmune thrombocytopenia.

The aim of this study was to investigate the relatively frequencies of alleles in the HLA-C*04:01:01G group and to analyze their relations with HLA-A and -B loci. DNA samples previously typed as HLA-C*04:01:01G were sequentially selected. The sequences for exon 2 to 7 of the HLA-C locus were analyzed by polymerase chain reaction sequence-based typing(PCR-SBT). The HLA-A, -B, -DRB1 and -DQB1 loci were genotyped using PCR-SBT method. The results showed that 178 samples (94.2%) and 11 samples (5.8%) were assigned as HLA-C*04:01:01 and HLA-C*04:82 respectively among 189 samples previously typed as HLA-C*04:01:01G. 72 haplotypes associated with HLA-C*04:01:01 and C*04:82 were found, in which the frequencies of 26 haplotypes were over 0.0050. HLA-C*04:01:01 was strongly related with A*02:03, A*02:07, A*11:01, A*33:03, B*13:01, B*15:01, B*15:05, B*15:27, B*40:01, B*54:01 alleles, while HLA-C*04:82 was related with B*40:01. It is concluded that HLA-C*04:01:01 and HLA-C*04:82 alleles were confirmed in the HLA-C*04:01:01G group, which should be discriminated by the routine HLA genotyping.

The individual with the deficiency of CD36 antigen on platelet displayed the risk of anti-CD36 immune reaction induced by transfusion, which is one of the reasons for platelet transfusion refractoriness (PTR). This study was purposed to detect the expression level of CD36 antigen on platelet by flow cytometry among apheresis platelet donors of Hangzhou area, and the frequency of CD36 deficiency was analyzed. Platelet-rich plasma (PRP) was separated from fresh anticoagulant whole blood by centrifugation, then the platelets were washed and adjusted to 1×10(6). The platelets were incubated with FITC-labeled CD36 and PE-labeled CD41 monoclonal antibodies, then the expression level of CD36 was detected by flow cytometry. The CD36 expression on monocytes for the samples of CD36-deficiency on the platelets was further analyzed. The results showed that 7 samples with CD36 antigen deficiency were found in 192 apheresis platelet donors. The frequency of CD36 deficiency was 3.6% and all of them were typeII deficiency. The significant difference of CD36 antigen expression was observed in the platelet donors of Hangzhou population, among them 59 individuals with low expressed CD36 antigen and 126 individuals with highly expressed CD36 antigen were found according to the geometric mean fluorescence intensity. It is concluded that the CD36 antigen deficient phenotype existed in the population, these data will provide the information for research of the CD36 antigen distribution and help to solve the platelet transfusion refractoriness.

This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.

This study was aimed to investigate the distribution of rare blood group in Zhejiang Han population. The H(-) (H system), GPA(-) and s(-) (MNS), Rhnull, Rhmod, D--, CCDEE, CCdEE (variations of Rh), GPC(-) (Gerbich), i(+) (I), Lu(b-) (Lutheran), Js(b-) and k(-) (Kell), Fy(a-) (Duffy), Ok(a-) (Ok), Di(b-) (Diego) phenotypes were screened by serological or molecular methods. Jk (a-b-) phenotype was detected by urea hemolytic test. The results showed that one Di (a+b-) individual was found in 1618 blood donors, three Fy (a-b+) individuals in 1007 donors and one CCdEE individual in 633 Rh negative donors. No Jk (a-b-), H(-), GPA(-), s(-), GPC(-), i(+) (adult), Lu(b-), k(-), Js(b-), Lu(b-) and Ok(a-) phenotypes were found in this large scale survey. It is concluded that Di (a+b-), Fy (a-b+), CCdEE phenotypes are confirmed in the blood donors and this study provides the distribution data of erythrocyte rare blood group in Zhejiang Han population.

This study was aimed to discriminate the alleles in the HLA-C*07:01:01G and HLA-C*07:02:01G groups and analyze their associations with HLA-B locus. Samples previously typed as HLA-C*07:01:01G and HLA-C*07:02:01G were collected. The nucleotide sequences in exons 1 to 7 of the HLA-C locus were sequenced by polymerase chain reaction sequence-based typing (PCR-SBT) and HLA-B genotyping was also preformed by PCR-SBT in these samples. The results showed that 4 samples (30.8%) were confirmed as HLA-C*07:01:01 and 9 samples (69.2%) were HLA-C*07:06 among 13 samples previously typed as HLA-C*07:01:01G. Linkage disequilibrium (LD) analysis showed that HLA-C*07:06 allele was strongly related with HLA-B*44:03. All samples were typed as C*07:02:01 among 102 individuals previously typed as C*07:02:01G. LD analysis showed that C*07:02:01 was strongly related with HLA-B*51:01, B*46:01, B*39:01, B*40:01, B*38:02, B*15:02 alleles. It is concluded that HLA-C*07:01:01 and HLA-C*07:06 alleles are confirmed in the HLA-C*07:01:01G group and HLA-C*07:02:01 is a preferred allele in the HLA-C*07:02:01G.

The objective of this study was to analyze the molecular genetic basis for 2 individuals with A2B phenotype of ABO subtype. The ABO group antigens on red blood cells were identified by monoclonal antibodies and the ABO antibodies in serum were detected by the standard A, B, O cells. The exon 5 to exon 7 coding region of ABO gene was amplified by polymerase chain reaction (PCR) and the PCR product was sequenced directly after the enzymes digested. The amplified product was also cloned by TOPO TA cloning sequencing kit to split the 2 alleles apart and chosen colonies were sequencing bidirectionally for exon 6 to 7 of ABO gene. The results showed that both A and B antigen were identified on red blood cells of the individuals and there was anti-A1 antibody in their serum. There was no 261G deletion and showed 297A/G, 467C/T, 526C/G, 657C/T, 703G/A, 742C/T, 796C/A, 803G/C, 930G/A heterozygotes by direct DNA sequencing. After cloning and sequencing, it was obtained the B101 and one novel A allele. The novel allele has one nucleotide change at 742 position C to T compared with A102, which results in an amino acid Arg to Cys at 248 position and was nominated as A213. It is concluded that C742T mutation of the α1, 3 N-acetyl-D-galactosaminyl-transferase gene can lead to A2 phenotype and with anti-A1 antibody in serum.

This study was purposed to investigate the molecular basis of a para-Bombay phenotype for screening and identification of rare blood group. ABO and H phenotypes of the proband were identified by serological techniques. The exon 6 to exon 7 of ABO gene and full coding region of α-1,2-fucosyltransferase (fut1) gene of the proband were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments. The haplotype of compound heterozygote of fut1 was also identified by cloning sequencing. The results indicated that a rare para-Bombay phenotype was confirmed by serological techniques. Two deletion or insertion variant sites near nucleotide 547 and 880 were detected in fut1 gene. The results of cloning sequence showed that one haplotype of fut1 gene was two bases deletion at 547-552 (AGAGAG→AGAG), and another one was two bases deletion at position 880-882 (TTT→T). Both two variants caused a reading frame shift and a premature stop codon. It is concluded that a rare para-Bombay phenotype is found and confirmed in blood donor population. The molecular basis of this individual is compound heterozygote of two bases deletion on fut1 gene which weaken the activity of α-1, 2-fucosyltransferase.

In order to explore the effects of 35C > T and 682A > G mutations on the activity of alpha-(1,2) fucosyltransferase, the coding region of fut1 gene was amplified by polymerase chain reaction (PCR) from genomic DNA. PCR product was ligated into expression vector using TOPO TA cloning kit to obtain the recombinant plasmids. The recombinant plasmids were transfected into COS-7 cells by liposome method. After screening by using G418, H antigen expression on the COS-7 was tested by flow cytometry and fut1 mRNA was detected by real-time PCR. The results indicated that three kinds of recombinant plasmids pcDNA3.1/V5-His-wild (35C + 682A), pcDNA3.1/V5-His-35T and pcDNA3.1/V5-His-35T-682G were successfully constructed. After transfection, the H antigen expressed on membrane of COS-7 cells at the second day, with the maximum level of expression at the fourth day. When compared with pcDNA3.1/V5-His-wild transfected cells, the H antigen expression level of the 35T and 682G + 35T recombinant plasmids in the transfected cells was 52.7% and 13.3% on the fourth day, respectively. Although the level of fut1 mRNA decreased with prolonging of time, the mRNA expressed on the pcDNA3.1/V5-His-35T-682G transfected cells reached to 14% of the wild plasmids on the first day. It is concluded that 682A > G mutation obviously reduces the activity of alpha-(1,2) fucosyltransferase, while 35C > T mutation leads to partial reduction of H antigen in vitro expression.

The aim of study was to explore the feasibility of quantitative chimerism analysis of regulatory T (Treg) cells using immune sorting coupling short tandem repeat (STR) method. 14 sets of artificial chimera samples were prepared by mixed lymphocytes according to different proportion. The CD4(+)CD25(+) Treg cells were harvested by negative and positive selection of immunomagnetic beads, then the STR polymorphisms of 16 loci in sorted Treg cells was analyzed. The results showed that the DNA amount extracted from sorted Treg cells was fit for STR detection. All STR alleles specific for recipient or donor could be detected and the quantitative results were consistent with theoretic values in over 10% recipient chimeras. But only partial recipient alleles could be detected and the quantitative results were different from theoretic values in less then 1% recipient chimeras. It is concluded that a quantitative chimerism analysis of Treg cell based on immune sorting is established. The sensitivity and accuracy for chimera detection are 1% to 10%, and this method can be used to monitoring hematopoietic chimerism following allogeneic hematopoietic stem cell transplantation.

This study was aimed to analyze the possibility of high resolution matching for human leukocyte antigen (HLA) loci in unrelated donor-recipient pair with low resolution match in HLA-A, -B, -DRB1 loci. Samples were genotyped for HLA-A, -B, -C, -DRB1 and -DQB1 by polymerase chain reaction sequence based typing (PCR-SBT). The results showed that the total number of patients and the donors were 166 and 274. 97 (58.43%) patients were matched for 1 donor and 47 (28.31%) patients were matched for 2 donors at low resolution level; among 274 donor-recipient pairs, HLA-A, -B, -C, -DRB1 and -DQB1 loci matching for 6/10, 7/10, 8/10, 9/10 and 10/10 were 32 (11.68%), 54 (19.71%), 62 (22.63%), 49 (17.88%) and 48 (17.52%) respectively; there were mismatch in HLA-A, -B, -C, -DRB1 and -DQB1 loci, and the most mismatch was in HLA-C locus. The number of alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci were 23, 46, 21, 30 and 17 respectively in the donors. The alleles number HLA-A, -B, -C, -DRB1 and -DQB1 loci were 20, 40, 22, 29 and 16 respectively in the patients; the haplotype number of HLA loci were 311 in the donors and 224 in the patients. The high frequency of haplotype was A*02:07-B*46:01-C*01:02-DRB1*09:01:02-DQB1*03:03 (5.63% and 6.88%). It is concluded that the probability of high resolution mismatch of HLA loci is high in unrelated donor-recipient pairs with low resolution match in HLA-A, -B, -DRB1 loci.

This study was purposed to investigate the nucleotide sequences of a novel HLA-B*15:124 allele and its molecular mechanism. The genomic DNA from whole blood was extracted by using commercial DNA extraction kit. The sequences of exon 2, 3 and 4 of HLA-B locus in the proband were amplified by PCR with group-specific primers, the PCR products were purified by enzymes digestion, then exon 2 to 4 of HLA-B locus for both orientations was sequenced. The results showed that 2 HLA-B alleles of proband were gained after amplification and sequencing of group-specific primers, among them one was a B*40:03, another was a novel allele. After BLAST analysis, the novel allele showed nucleotides different from HLA-B*15:52 in exon 3 at nucleotide position 427 A > T and 440 G > T which resulted in amino acid change from Thr to Ser at codon 143 and Trp to Leu at conon 147. It is concluded that a novel HLA-B allele has two different nucleotides. This HLA-B allele is identified and has been officially named B*15:124 by the WHO Nomenclature Committee.

In order to construct prokaryotic expression system of MHC classI chain-related gene A (mica) and purify MICA protein, RNAs were extracted from the peripheral blood samples and mica cDNA fragments were obtained by RT-PCR method. The cDNA for mica was ligated with cloning vector by TOPO method. The recombinant cloning vector and prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct pET-28a-MICA recombinant expression vector, then the pET-28a-MICA vector was transformed and expressed in E. coli BL21 DE3. The recombinant protein was purified by Ni-NTA Spin method. The results showed that the recombinant MICA protein expressed with soluble form in host with pET-28a-MICA vector after IPTG induction. The recombinant target protein was obtained by Ni-NTA spin purification. In conclusion, this study has constructed prokaryotic expression system of mica gene and has purified MICA protein which would help to explore the interaction between MICA and transplantation immunology.

Objective of this study was to explore the molecular basis of a new O61 allele in ABO blood group. The ABO group antigens on red cells of the blood samples were identified by monoclonal antibodies and the ABO antibody in serum was detected by the standard A, B, O red cells. The coding region sequences of exon 5 to exon 7 in ABO gene were amplified by polymerase chain reaction (PCR) and the amplification products were purified with double enzyme digestion and directly sequenced for exon 6 and 7. The diploid of the individual with B phenotype was separated into its haploid components with a haplotype specific extraction method. The exons 6 to 7 of the two single ABO haplotypes were then amplified and sequenced separately. The results indicated that 3 samples had mutation at 743 site in total 417 individuals, in which 2 individuals were with O phenotype and 1 individual was with B phenotype. The DNA sequencing of exon 6 and 7 in 2 samples with O phenotype showed 261G deletion and 743G/C heterozygotes. The DNA sequencing of exon 6 and 7 in the sample with B phenotype showed 261G/deletion and 297A/G, 526C/G, 743G/C, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A heterozygotes. After separating of the 2 single strands in the B sample with haplotype specific extraction, an O and B101 allele were identified after sequencing. The novel allele was submitted to the Blood Group Antigen Gene Mutation database and is named as O61. It is concluded that 743G>C is a novel mutation in exon 7 of ABO and a novel O61 allele with 743G>C has been identified.

This study was aimed to investigate the changes of cytokine contents in single donor platelets (SDPs) collected by using MCS(+), Trima, Amicus blood cell separators during storage. 18 portions of SDPs were collected by MCS(+), Trima, Amicus blood cell separators, were preserved in standard condition of blood bank, the levels of cytokines such as IL-8, RANTES, CD154, TGF-beta(1) and VEGF were detected by ELISA at 1, 3, 5, 7 days during storage. The results showed that the levels of IL-8, RANTES, CD154, TGF-beta(1) and VEGF in SDPs collected by blood cell separators MCS(+), Trima, and Amicus gradually increased with prolonging of time during storage, but the increase of IL-8 level in SDPs collected by MCS(+) separator was significant difference from SDPs collected by Trime and Amicus separators (p < 0.05). It is concluded that the all collected SDPs mentioned above express IL-8, RANTES, CD154, TGF-beta(1) and VEGF during storage, and their cytokine levels show a tendency to increase with prolonging of time during storage, apheresis platelets with less leukocytes express IL-8 lower.